Arginine deiminase (EC 3.5.3.6), abbreviated as ADI, can hydrolyze arginine to produce citrulline and ammonia. Since the first report in 1933, the enzyme has been found in Streptococcus lactiae, Streptococcus faecalis, yeast, pseudomonas, mycoplasma, halobacterium and some eukaryotic cells, etc. Arginine deaminase is wide in source, and has been found in bacteria, archaebacteria and some eukaryotic cells. There are obvious differences in properties of ADI of different microbial sources, such as molecular weight range, optimal pH, optimal temperature, etc. However, all the ADI has the ability to decompose arginine.
Citrulline, also called as carbamylornithine ornithin, gets the name as being extracted from watermelon initially. In recent years, it has been found by researches that citrulline has the physiological functions of scavenging free radicals, affecting nitrogen balance in the human body, immune regulation of the cardiovascular system, and the like. Therefore, the application of citrulline in food and medicine aspects is increasing. Because of the functions of aging resisting, immunity enhancement, athlete muscle strength and endurance improvement and the like of citrulline, citrulline has been added into sports functional drinks produced by a Canadian company. At the same time, health skin care products containing citrulline are also sold on the market in European and American countries.
Citrulline is mainly produced by four methods, i.e., a chemical method, an extraction method, a fermentation method and an enzymic method. Among them, the chemical method is currently the main production mode, but by-products such as copper ions and hydrogen sulfide produced during a production process have an adverse impact on the environment. Both the extraction method and the fermentation method for citrulline production have the of low yield and high cost, and thus are not conducive to large-scale production. On the contrary, the enzymatic method for citrulline production has the advantages of mild reaction conditions, high conversion efficiency, simple extraction process and the like, so the enzymatic method has high research and production value. Therefore, the search for safe and stable arginine deiminase with high catalytic efficiency is the key issue for enzymatic citrulline production.
Site-directed mutagenesis as a major means of molecular transformation refers to a technology of introducing a specific base pair at a designated site of a target DNA fragment to change an amino acid sequence encoded by the Site-directed mutagenesis is more rapid, direct and cost-effective than other strategies for improving a molecular structure, so it is one of the most commonly used genetic modification means in laboratories.